APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1976, p. Copyright C 1976 American Society for Microbiology

433-438

Vol.

31,

No. 3

Printed in USA.

NOTES Double-Stranded Ribonucleic Acid in Agaricus bisporus R. MARINO, K. N. SAKSENA, M. SCHULER, J. E. MAYFIELD, AND P. A. LEMKE* Carnegie-Mellon Institute of Research and the Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213

Received for publication 29 August 1975

Double-stranded ribonucleic acid present in virus-infected mushrooms of Agaricus bisporus has been resolved through polyacrylamide gel electrophoresis into six molecular-weight forms. Identification of these six double-stranded ribonucleic acids in mushrooms by this procedure has proven to be a useful and diagnostic method for viral infection in the cultivated mushroom. Viruses containing double-stranded ribonucleic acid (dsRNA) are ubiquitous in fungi and, with few exceptions, constitute latent infections (7, 16). One apparent exception is a disease of the cultivated mushroom, Agaricus bisporus, first described in 1950 (13) and subsequently shown to be related to the presence of virus-like particles of at least three types (1, 2, 4, 5, 11).

Nucleic acids extracted from mushrooms of A. bisporus (strain D-26) have been examined for dsRNA content by polyacrylamide gel electrophoresis. Procedures for the extraction and electrophoretic analysis of nucleic acids from these mushrooms followed basically the procedures outlined by Vodkin and co-workers for analysis of dsRNA from yeast (15). However, gels contained 7.5% acrylamide, 0.187% N,N'-methylene-bisacrylamide, 0.11% TEMED (N,N,N',N'-tetramethylethylenediamine), and 0.03% ammonium persulfate. Gels 8 to 10 cm long were run from 5 to 10 h at 2.5 mA/gel and stained with either 0.2% methylene blue or 0.01% toluidine blue. Six electrophoretic bands (Fig. 1A) have consistently been identified among nucleic acids extracted from diseased mushrooms containing virus particles of three morphological types (11). These particles measure approximately 26 (spherical), 35 (spherical), and 51 by 20 nm (bacilliform). Normal-appearing mushrooms of strain D-26, whether taken from cultivation or grown in the laboratory under controlled conditions (12), generally lack virus particles and the six electrophoretic bands routinely associated with diseased mushrooms. Evidence for the double-stranded and RNA nature of the six bands revealed by electropho-

resis includes the following. (i) Analytical ultracentrifugation of nucleic acid samples from virus-infected mushrooms according to procedures outlined by Szybalski (14) shows a major component with a buoyant density in cesium sulfate of about 1.60 g/cm3, an expected density value for double-helical RNA. This component is absent from nucleic acids derived from normal mushrooms (Fig. 2). (ii) In heat-treated (100 C for 5 min) samples of nucleic acid from virus-infected mushrooms, the material of 1.60 g/cm3 buoyant density apparently melts into single-stranded RNA, as evidenced by a shift in buoyant density to 1.62 g/cm3 (Fig. 3). Electrophoretic analysis of heat-treated samples also indicates melting, with loss of the six bands and the appearance of a broad band in gels with a migration indicative of high-molecular-weight single-stranded RNA (Fig. 4). (iii) The electrophoretic behavior of the six bands under consideration is consistent with that of known dsRNA's insofar as they migrate as sharp bands (Fig. 5) and stain violaceous-pink in toluidine blue. Coelectrophoresis of nucleic acids from mushrooms and standard reovirus (type 3) dsRNA (Fig. 1B) indicates that the six electrophoretic bands unique to virus-infected mushrooms migrate within the molecular weight range of reovirus dsRNA (3). (iv) All six electrophoretic bands were resistant to treatment with deoxyribonuclease (type I, 15 ,ug/ml; Sigma) but showed only relative resistance to treatment with bovine pancreatic ribonuclease (type IIA, 4 ,ug/ml; Sigma). This was indicated by treating nucleic acid samples during extraction with deoxyribonuclease (25 C, 30 min) in 0.1 M sodium acetate containing 0.005 M MgSO4 (pH 5.0) and with ribonuclease (37 C, 30 min) in 433

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FIG. 1. (A) Acrylamide gel electrophoresis of nucleic acid samples from virus-infected mushrooms (left), normal mushrooms (center), and standard reovirus type-3 dsRNA (right). Gels 10 cm in length were run for 10 h at 2.5 mA/gel and stained with methylene blue. (B) Migration of Agaricus dsRNA relative to reovirus dsRNA. Gels were run for 9 h at 2.5 mA/gel and scanned with a Beckmann model 24/25 gel scanner at optical density at 260 nm. Assuming linear logarithmic migration of dsRNA based on molecular weight, for the range defined by reovirus dsRNA (3), then the six dsRNA's from mushrooms would have approximate molecular weights of 2.17, 1.89, 1.76, 1.70, 1.60, and 0.67 X 106.

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i p=lt.482

1.605

FIG. 2. Analytical ultracentrifugation (Beckmodel E, 44,000 rpm, 68 h) in cesium sulfate of nucleic acid samples derived from virus-infected (top) and normal (bottom) mushrooms. A prominent band with a buoyant density (p) of approximately 1.60 g/cm: appears only in the sample from diseased mushrooms.

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VOL. 31, 1976

NOTES

p

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435

=1.598

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p= 1.479

HEATED

FIG. 3. Analytical ultracentrifugation (44,000 rpm, 48 h) in cesium sulfate of an unheated (top) and heated (bottom) nucleic acid sample derived from virus-infected mushrooms. An apparent shift in buoyant density (p = 1.60 glcm3 -+ 1.62 g/cm3) is evident in the heat-treated (100 C, 5 min) material. Absorption profiles were recorded with a Joyce-Loebl densitometer.

436

APPL. ENVIRON. MICROBIOL.

NOTES

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FIG. 4. Electrophoresis of an unheated (left) and heated (right) nucleic acid sample derived from virusinfected mushrooms. Gels were run for 5 h at 2.5 mA/gel and stained with methylene blue. Absorption profiles (optical density at 260 nm) of unstained gels confirm results from staining and indicate thermal melting of six bands present in the unheated sample.

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FIG. 5. Electrophoretic separation of six bands in a nucleic acid sample derived from virus-infected mushrooms. This gel was run for 10 h at 2.5 mA and stained with toluidine blue. Electrophoretic profile was recorded at an optical density at 260 nm prior to staining.

VOL. 31, 1976

either standard saline citrate (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) or dilute SSC (0.1 SSC). The six bands were hydrolyzed only after ribonuclease treatment in the dilute SSC, as evidenced by their disappearance from gels (Fig. 6). (v) Finally, some component(s) present in nucleic acids from virus-infected mushrooms, but not in nucleic acids from normal mushrooms, reacts positively with antiserum prepared to a synthetic dsRNA [poly(I):poly(C)] (Fig. 7). Serological cross-reactivity of natural dsRNA to anti-poly(I):poly(C) has been useful in detecting dsRNA viruses in other fungi (8, 9). Studies are in progress to determine association of dsRNA's from mushrooms with specific particle types. Preliminary information has FIG. 6. Nuclease treatment of nucleic acid samples derived from virus-infected mushrooms. Evidence for deoxyribonuclease (DNase) resistance of the six electrophoretic bands is indicated by the gel on the left. Resistance to ribonuclease (RNase) treatment at a salt concentration of standard saline citrate (SSC) is indicated by the center gel. All six bands, however, are lost (right) from the sample treated with RNase at low salt concentration (0.1 SSC). Gels were run for 9 h at 2.5 mA/gel and stained with methylene blue.

NOTES

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FIG. 7. Agar double-diffusion test for dsRNA using antiserum (center well) to poly(I):poly(C). (A) and (C) contain nucleic acid samples derived from virus-infected mushrooms; (B) contains authentic dsRNA from Penicillium chrysogenum (7); (D) contains nucleic acid sample derived from normal mushrooms.

438

APPL. ENVIRON. MICROBIOL.

NOTES

been published (6, 10) that indicates that dsRNA is present in virus particles ofA. bisporus with spherical morphology rather than in particles of the bacilliform type. This research was supported by the Butler County Mushroom Farm, Inc. of Worthington, Pa., through a fellowship (1-41010) to the Carnegie-Mellon Institute of Research. We are grateful to A. Shatkin for a sample of reovirus dsRNA and R. M. Lister for antiserum to dsRNA.

6. Lapierre, H., G. Molin, C. Kusiak, and A. A. Faivre. 1973. L'acide nucleique des virus de champignons. Ann. Phytopathol. 5:322-325. 7. Lemke, P. A., and C. H. Nash. 1974. Fungal viruses. Bacteriol. Rev. 38:29-56. 8. Moffitt, E. M., and R. M. Lister. 1974. Detection of mycoviruses using antiserum specific for ds-RNA. Virology 52:301-304. 9. Moffitt, E. M., and R. M. Lister. 1975. Application of a serological screening test for detecting double10.

1.

2.

3. 4. 5.

LITERATURE CITED Dieleman-van Zaayen, A. 1972. Mushroom virus disease in the Netherlands: symptoms, etiology, electron microscopy, spread and control. Centre for Agricultural Publishing and Documentation, Wageningen, Netherlands. Dieleman-van Zaayen, A., and J. H. M. Temmink. 1968. A virus disease of cultivated mushrooms in the Netherlands. Neth. J. Plant Pathol. 74:48-51. Fujii-Kawata, I., K. Miura, and M. Fuke. 1970. Segments of genome of viruses containing doublestranded ribonucleic acid. J. Mol. Biol. 51:247-253. Hollings, M. 1962. Viruses associated with a die-back disease of cultivated mushroom. Nature (London) 169:692-695. Hollings, M., D. G. Gandy, and F. T. Last. 1963. A virus disease of a fungus: die-back of cultivated mushroom. Endeavor 22:112-117.

11. 12. 13. 14.

15. 16.

stranded RNA mycoviruses. Phytopathology 65:851859. Molin, G., and H. Lapierre. 1973. L'acide nucl6ique des virus de champignons: cas de virus de l'Agaricus bisporus. Ann. Phytopathol. 5:233-240. Saksena, K. N. 1975. Isolation and large-scale purification of mushroom viruses. Dev. Ind. Microbiol. 16:134-144. San Antonio, J. P. 1971. A laboratory method to obtain fruit from cased grain spawn of the cultivated mushroom, Agaricus bisporus. Mycologia 63:16-21. Sinden, J. W., and E. Hauser. 1950. Report on two new mushroom diseases. Mushroom Sci. 1:96-100. Szybalski, W. 1968. Use of cesium sulfate for equilibrium density gradient centrifugation, p. 330-360. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12B. Academic Press Inc., New York. Vodkin, M., F. Katterman, and G. R. Fink. 1974. Yeast killer mutants with altered double-stranded ribonucleic acid. J. Bacteriol. 117:681-686. Wood, H. A. 1973. Viruses with double-stranded RNA genomes. J. Gen. Virol. 20:61-85.